Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

Ex vivo lung perfusion, or EVLP, enables extended evaluation of donor lungs. There are two perfusion solutions, an acellular and cellular. This study details using a polymerized human hemoglobin, or PolyHb, based oxygen carrier as a perfusate in the protocol for testing this solution in a rat EVLP model.

The recent advances in ex vivo organ perfusion have enabled expanding the donor organ pool with improved function. Working to extend normothermic perfusion times with tailored perfusates will allow for further advances in this space. Current perfusion techniques use cellular or acellular perfusates, each with pros and cons.

Perfusion composition is crucial for metabolic support, reducing inflammation, and maintaining oncotic pressure. However, existing solutions can cause pulmonary edema and damage, thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis. A promising hemoglobin-based oxygen carrier, or HBOC, is polymerized Human hemoglobin synthesized from expired packed red blood cells.

Our laboratory developed a next-generation PolyHb with minimal low molecular weight species and cell-free hemoglobin. This has shown improved biophysical characteristics and has reduced vasoconstriction, hypertension, and oxidative damage in animal studies. These characteristics make it a promising perfusate candidate.

To begin, take human serum albumin at a final concentration of 3%Into it, add polymerized hemoglobin, or PolyhHb, diluted to a final concentration of 3.7 grams per deciliter with Williams'E Medium. Then, add one milliliter of heparin solution to the resultant diluted PolyHb solution. Place PolyHb perfusate into the EVLP circuit reservoir and set the water bath temperature to 37 degrees Celsius.

Then, turn on the roller pumps and ensure that perfusate is circulating within the circuit. Connect deoxygenation gas to the hollow fiber oxygenator to deoxygenate the perfusate. Open data acquisition software on the computer.

Verify that pulmonary artery pressure, tracheal differential pressure, respiratory flow differential pressure, lung weight, and pump speed transducers are connected to both the circuit and data converter box. Carefully examine all tube connections to ensure no leaks are present and warm water is circulating throughout. Press run on the software to check that all pressure transducers are functioning.

Once the system is properly functioning, turn off the roller pumps. To begin, place the autoclave surgical instruments on the surgical table. Shave the abdomen of the anesthetized rat and place the rat in the supine position on the surgical board.

Clean the abdomen with povidone iodine followed by 70%ethanol. Place ophthalmic ointment under the rat's eyes to prevent dryness. Secure the rat in place on the surgical board.

Start the data acquisition software and begin recording. Turn on the ventilator at four milliliters per kilogram and ensure positive and expiratory pressure is around two centimeters of water. Using scissors, perform a midline laparotomy from the xiphoid process to the pubic synthesis.

Then, with the help of a blunt instrument, perform a medial lateral visceral rotation and visualize the infrahepatic inferior vena cava. Inject heparin into the inferior vena cava with a 20-gauge needle. Using a pair of scissors, cut the skin from the sternal notch to just below the angle of the mandible and begin to dissect toward the trachea.

Then, bluntly dissect away necessary strap muscles to expose the trachea. Make a transverse incision on the anterior trachea between the cartilaginous rings, ensuring not to cut through the posterior portion of the trachea. Place a 5-0 silk suture around the trachea.

Insert the endotracheal tube into the cartilaginous rings and secure it with the 5-0 silk suture. Connect the endotracheal tube to the ventilator and ensure proper chest rising. Using scissors, perform a median sternotomy and enter the thoracic cavity.

Place chest wall retractors to expose the heart and lungs, avoiding any inadvertent manipulation of the lungs. With a combination of sharp and blunt dissection, remove the thymus from the anterior mediastinum. Identify the pulmonary artery and place a 5-0 silk suture around it to prepare for cannulation.

Make a two to three millimeter incision in the right ventricular outflow tract using scissors to place the arterial cannula within the pulmonary artery and secure it with the 5-0 suture. After euthanizing the rat, quickly connect the deaired lung preservation fluid to the arterial cannula to gravity flush the lungs. Connect the arterial cannula to the EVLP circuit.

Turn on the roller pump and allow a small amount of perfusate to flow through the lung and out of the left ventricle into the thoracic cavity. Once perfusate begins to flow out of the left atrium, turn off the roller pump. Next, place a small forceps in the left ventricle and gently stretch the mitral valve annulus.

Place a 5-0 silk tie around the heart and loosely tie. Insert the left atrium cannula into the left ventricle and advance the cannula until it can be seen within the atrium. Secure the left atrium with the pre-tied 5-0 suture.

Identify the esophagus and clamp it with a hemostat as close to the diaphragm as possible. Then, cut the esophagus below the hemostat. Using the spine as a guide, cut all ligamentous attachments connecting the heart-lung block to the surrounding structures with scissors.

Then, dissect the trachea from the neck and cut the trachea above the endotracheal tube to free the heart-lung block. Move the heart-lung block to the thoracic jacket within the EVLP circuit and attach the left atrium cannula to the circuit. Turn the roller pump on and connect the ventilator monitor.

Check the bubble trap to ensure no air emboli are being introduced into the system. Slowly change ventilation and perfusion settings to the desired experimental levels during the initial 15 minutes. Additionally, increase the perfusion flow rate to the desired rate and pressure.

At designated time points, check perfusate gas levels and pulmonary function tests. All tested perfusates showed a slight decrease in left atrium partial pressure of oxygen, with the RBC-based perfusate significantly decreasing at one hour. For the next several hours, both PolyHb and control perfusates had stable left atrium partial pressure of oxygen.

The delta partial pressure of oxygen significantly decreased at one hour in the RBC perfusate group, while it remains stable in the PolyhHb and control perfusates with a non-significant trend. Left atrium partial pressure of carbon dioxide was significantly lower in the RBC and control perfusate compared to PolyhHb after the first hour, and this trend continues over the following hours. The delta partial pressure of carbon dioxide was significantly increased in the RBC perfusate after one hour, and after that, remains stable in both the PolyhHb and control perfusate.

Realtime lung physiological data demonstrate that the RBC perfusate significantly increased pulmonary vascular resistance within the first hour, while both PolyhHb and control perfusates maintained low and stable pulmonary vascular resistance over the period. The change in lung weight was significant in the RBC perfusate initially, with a continued increase in all perfusates, slightly more in PolyhHb. Compliance decreased significantly in the RBC perfusate within the first hour, while it decreased non-significantly in other perfusates.

PolyhHb had the highest compliance after four hours.